phospholipases A
2Timo J. Nevalainen1, Joa˜o C. R. Cardoso2and Pentti T. Riikonen3
1 Department of Pathology, University of Turku and Turku University Hospital, Finland
2 Comparative Molecular Endocrinology, Centre of Marine Sciences, Universidade do Algarve, Campus de Gambelas, Faro, Portugal 3 Department of Information Technology, University of Turku, Finland
Keywords
conserved domains; eukaryotes; evolution; prokaryotes; secreted PLA2s
Correspondence
Timo J. Nevalainen, Department of Pathology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland
Fax: +358 2 3337456 Tel: +358 2 3337500 E-mail: timneva@utu.fi
(Received 1 September 2011, revised 8 December 2011, accepted 13 December 2011)
doi:10.1111/j.1742-4658.2011.08453.x
Secreted phospholipases A2 (sPLA2s) are lipolytic enzymes present in organisms ranging from prokaryotes to eukaryotes but their origin and emergence are poorly understood. We identified and compared the con-served domains of 333 sPLA2s and proposed a model for their evolution. The conserved domains were grouped into seven categories according to the in silico annotated conserved domain collections of ‘cd00618: PLA2_ like’ and ‘pfam00068: Phospholip_A2_1’. PLA2s containing the conserved domain cd04706 (plant-specific PLA2) are present in bacteria and plants. Metazoan PLA2s of the group (G) I⁄ II ⁄ V ⁄ X PLA2 collection exclusively contain the conserved domain cd00125. GIII PLA2s of both vertebrates and invertebrates contain the conserved domain cd04704 (bee venom-like PLA2), and mammalian GIII PLA2s also contain the conserved domain cd04705 (similar to human GIII PLA2). The sPLA2s of bacteria, fungi and marine invertebrates contain the conserved domain pfam09056 (prokaryotic PLA2) that is the only conserved domain identified in fungal sPLA2s. Pfam06951 (GXII PLA2) is present in bacteria and is widely distributed in eukaryotes. All conserved domains were present across mammalian sPLA2s, with the exception of cd04706 and pfam09056. Notably, no sPLA2s were found in Archaea. Phylogenetic analysis of sPLA2 conserved domains reveals that two main clades, the cd- and the pfam-collection, exist, and that they have evolved via gene-duplication and gene-deletion events. These observations are consistent with the hypothesis that sPLA2s in eukaryotes shared common origins with two types of bacterial sPLA2s, and their persistence during evolution may be related to their role in phos-pholipid metabolism, which is fundamental for survival.
Introduction
Phospholipases A2
Phospholipases A2 (PLA2s; EC 3.1.1.4) are a group of lipolytic enzymes that hydrolyze the sn-2 bond of phospholipids, such as phosphatidylcholine and phos-phatidylethanolamine, resulting in the release of fatty acid and lysophospholipid. They have been isolated
from organisms ranging from bacteria to mammals and are suggested to have emerged early in evolution. In general, PLA2s are classified into three broad cate-gories: Ca2+-dependent secreted PLA2s (sPLA2s); Ca2+-dependent cytosolic PLA2s; and Ca2+ -indepen-dent cytosolic PLA2s. Cytosolic PLA2s are known to play an important role in cellular signalling and
Abbreviations
EST, expressed sequence tag; G, group; ML, maximum likelihood; NJ, neighbour-joining; OtoC, N-terminal region of otoconin; OtoN, N-terminal region of otoconin; PLA2,phospholipase A2;sPLA2,secreted phospholipase A2.
prostanoid metabolism [1,2]. Secreted PLA2s are com-ponents of various body fluids, including blood plasma, pancreatic juice, tears, seminal fluid, and snake and other venoms, and they participate in diverse physiological and pathological functions, such as in the digestion of dietary phospholipids, in inflammatory reactions and in the defence against bacteria and other pathogens [1–7].
Secreted PLA2s
Secreted PLA2s are the product of distinct genes and they have been classified, according to their molecular structure, into the following groups: IA, IB, IIA, IIB, IIC, IID, IIE, IIF, III, V, IX, X, XIA, XIB, XIIA, XIIB, XIII and XIV [1]. Evidence of structural similar-ity among group (G)I, GII, GV and GX members was taken to suggest that they may form a distinct GI⁄ II ⁄ V ⁄ X PLA2 collection [8]. The GXIV PLA2s of bacteria and fungi differ in their primary structure and folding from the other sPLA2s [1].
Secreted PLA2s are small-molecular-mass proteins (14-18 kDa, 120-135 amino acid residues) that require the presence of Ca2+ at millimolar concentrations for their catalytic activity. They share a conserved 3D structure that is stabilized by five to eight disulfide bonds. The structure of the GI, GII and GX PLA2s consists of three a-helices, a two-stranded b-sheet (the b-wing) and a conserved Ca2+-binding loop [9–13]. The conserved catalytic network of GIB and GIIA PLA2s consists of hydrogen-bonded side-chains formed by histidine, which is localized in the long a-helix1, tyrosine and aspartic acid residues, as well as the hydrophobic wall that shields it [14]. The sPLA2s of plants have a different 3D structure. In the GXIB PLA2 of rice (Oryza sativa) the b-wing is absent and the C-terminal a-helix3 has a different ori-entation [15]. The sPLA2s of prokaryotes and fungi are characterized by a dominant a-helical fold [16]. The 3D structure of GIII PLA2 differs from that of GI⁄ II ⁄ V ⁄ X PLA2s but they share identical motifs [17].
Conserved domains of sPLA2s and their evolution Functional motifs of sPLA2s that are well conserved include the Ca2+-binding and catalytic sites, and the conserved cysteine residues and disulfide bond pat-tern [1,2]. In silico database annotations identified two conserved signature patterns: the ‘PA2_HIS (PS0018, Phospholipase A2 histidine active site C-C-{P}-x-H-{LGY}-x-C’, where x represents a nonconserved amino acid, and amino acids within brackets are not
allowed) that contains the histidine residue of the sPLA2 active site, and the ‘PA2_ASP (PS00119, Phospholipase A2 aspartic acid active site [LIVMA]-C-{LIVMFYWPCST}-C-D-{GS}-{G}-{N}-x-{QS}-C’, where x represents a nonconserved amino acid, amino acids within curly brackets are not allowed and amino acids within square brackets are allowed) centred on the active site aspartic acid residue and localized towards the C-terminal portion of the mole-cule [18]. The Ca2+-binding motif, which, for exam-ple in mammalian GIB sPLA2s, is Y-x-G-x-G (where x represents a nonconserved amino acid) is localized before the histidine catalytic site towards the N-ter-minus.
Studies of sPLA2s have focussed mainly on eukary-otes [1–4]. However, the availability of molecular data from phylogenetically distant organisms, and the iden-tification of new sPLA2s, challenges the current classi-fication system, especially when applied to invertebrate and prokaryote sPLA2s [1,19–21]. Despite their poten-tial evolutionary relationships [22,23], the origin and emergence of sPLA2s is still intriguing. Although all sPLA2s share the same catalytic mechanism involving the canonical histidine residue, there is considerable variation in their sequence identity. Based on their identity, members of the GI⁄ II ⁄ V ⁄ X PLA2 collection have been designated ‘conventional sPLA2s’ and are believed to be evolutionally close, whereas GIII and GXII PLA2s are classified as ‘atypical sPLA2s’ and are evolutionally more distant [4,8]. In bacteria and fungi there are sPLA2s that show only limited sequence iden-tity with other sPLA2s [1,16], and to date the evolu-tionary connection between these structurally distant proteins is lacking.
The present study aimed to contribute to the under-standing of the origin and evolution of sPLA2s based upon the identification of their conserved domains in a representative collection of prokaryotes and eukary-otes. Recently, based on published data and the collec-tion of well-annotated multiple sequence alignment models of their conserved domains, the family hierar-chy of sPLA2s was comprehensively classified in two general collections: ‘cd00618 PLA2_like: Phospholipase A2, a superfamily of secretory and cytosolic phospho-lipases A2’ and ‘pfam00068: Phospholip_A2_1, Phos-pholipase A2’ [24,25]. In the present study, secreted PLA2 protein sequences retrieved from public databas-es were classified according to their conserved domain structures. Their sequences were compared and specific motifs within each subfamily group were identified, and, based upon their sequence and structure similar-ity, a model for their origin and evolution was proposed.
Results
Repertoire of prokaryotic and eukaryotic sPLA2s A total of 333 sPLA2 sequences from a wide range of taxa were retrieved from publicly available databases, and 358 conserved domains within the cd00618 (PLA2 _-like) and pfam00068 (Phospholip_A2_1) collections were identified (Tables 1 and S1). Members of the cd-collection identified are grouped into five distinct sub-families: the ‘cd04706 PLA2_plant: Plant-specific sub-family of Phospholipase A2’; ‘cd00125 PLA2c: Phospholipase A2, a family of secretory and cytosolic enzymes’; ‘cd04707 otoconin_90: Phospholipase A2-like domains present in otoconin-90 and otoconin-95’; ‘cd04704 PLA2_bee_venom_like: A subfamily of Phos-pholipase A2, similar to bee venom PLA2’ and ‘cd04705 PLA2_group_III_like: A subfamily of Phospholipase A2, similar to human group III PLA2’. The pfam-collec-tion members are subdivided into ‘pfam06951: PLA2G12, group XII secretory phospholipase A2 pre-cursor’ and ‘pfam09056 Phospholip_A2_3: Prokaryotic phospholipase A2 domain found in PLA2s of bacteria and fungi’ [24,25, http://www.ncbi.nlm.nih.gov/cdd].
All conserved domains of sPLA2s identified are present in representatives of the Kingdom Animalia with the exception of cd04706 of the sPLA2s of bacteria and plants (Table 1). Within this Kingdom, sPLA2s containing the conserved domain cd00125 are widespread in organisms ranging from basal metazoa (Porifera, Placozoa, Cnidaria and Rotifera), protosto-mes (Insects, Nematodes, Molluscs and Arthropods) and early deuterostomes (Echinodermata, Cephalocor-data and Tunicata) to teleosts and tetrapods (Amphi-bia, Aves and Mammalia). The conserved domain cd00125 is typical of GIA, IB, IIA, IIB, IIC, IID, IIE, IIF, V and X sPLA2s. The conserved domain cd04707 was found in the N- and C-terminal regions of vertebrate otconins. The conserved domains
cd04704 and cd04705 are present in metazoan GIII PLA2s, the latter domain exclusively in mammals. The sPLA2s of bacteria contain either cd04706 or pfam09056 (present in XIV PLA2s) and exceptionally pfam06951. In contrast, no sPLA2s were found in archaea. Pfam09056 is present in the sPLA2s of fungi. The majority of plant sPLA2s contained the con-served domain cd04706 (identified in XIA and XIB PLA2s). The conserved domain pfam06951 is present in the GXIIA PLA2s of unicellular and multicellular organisms, including marine algae and bacteria (Tables 1 and S1).
Conserved domain cd04706 of PLA2s of bacteria and plants
The sPLA2s of bacteria and plants share a number of conserved structural features for the histidine and aspartic acid catalytic motifs and Ca2+-binding domains (Fig. 1). The conserved domain cd04706 was identified in the sPLA2s of bacteria of the class Alphaproteobacteria of the phylum Proteobacteria and also in those of the phylum Firmicutes, which includes both Gram-negative and Gram-positive bacteria, such as the human pathogens Streptococ-cus pyogenes, Clostridium perfringens, Clostridium bot-ulinum and Bacillus cereus [26] (Table S1). Secreted PLA2s containing cd04706 were also identified in numerous plants. The sPLA2s of O. sativa are well characterized and classified as GXI PLA2s [15,27]. In general, several GXI PLA2 isoforms were identified in plants, which may have resulted from gene-dupli-cation events, for example, four GXI PLA2s were identified in O. sativa (Table S1). The GXIB PLA2 of O. sativa is a 16.6 kDa protein in which the Ca2+-binding site contains tyrosine, glycine and aspartic acid residues, and the histidine residue of the active site is centred in the catalytic site motif (Fig. 1A).
Table 1. Distribution of the conserved domains of sPLA2in the Kingdoms of Life. +, presence;), absence.
PLA2conserved domain Group numbera Archaea Bacteria Viridiplantae Fungi Animalia
cd00125 IA, IB, IIA, IIB, IIC, IID, IIE, IIF, V, X ) ) ) ) +
cd04704 III ) ) ) ) +
cd04705b III ) ) ) ) +
cd04706 XIA, XIB ) + + ) )
cd04707 Otoconin ) ) ) ) +
pfam06951 XIIA, XIIB ) + + + +
pfam09056 XIV ) + ) + +
aGroup number according to the classification in Schaloske and Dennis [1].bcd04705 is found only in mammalian GIII PLA 2s.
Sequence comparisons revealed that the plant (O. sa-tiva) and bacterial (S. pyogenes) sPLA2s share 17% amino acid sequence identity. In both proteins, there is an asparagine residue in the C-terminal portion of the molecule, which probably contributes to the catalytic function [15], instead of the more commonly occurring aspartic acid residue. In the GXIB PLA2 of O. sativa there are 12 cysteine residues that form six disulfide bonds [15], while the conserved domain of S. pyogenes sPLA2 contains five cysteine residues, four of which (C1-C2 and C3-C5), align with the plant GXIB PLA2 cysteines (C4-C8and C9-C10) and form conserved disul-fide bonds, and are suggestive of structural similarity (Fig. 1B).
Conserved domain cd00125 of PLA2of animals The conserved domain cd00125 was identified in the basal invertebrates Placozoa up to mammals and was prevalent in the vertebrate GI⁄ II ⁄ V ⁄ X PLA2 collec-tion where the funccollec-tional sites and cysteine residues are highly conserved (Fig. 2). Members of this collec-tion share 26–50% amino acid sequence identity within the conserved domain region. Among the members of the GI⁄ II ⁄ V ⁄ X PLA2 collection, the cata-lytic histidine is centred in the active site motif and
the aspartic acid residue of the active site is present in the C-terminal portion of the molecule. In the Ca2+-binding site, Ca2+ is bound by tyrosine and two glycines and an aspartic acid adjacent to the catalytic histidine (Fig. 2).
Homologues of GI⁄ II ⁄ V ⁄ X PLA2 collection mem-bers are present also in invertebrate genomes, such as the sea anemone Nematostella vectensis, where six genes were identified (Table S1) [28]. The sPLA2 of the sea anemone Adamsia carciniopados (also called Adam-sia palliata) contains the pancreatic loop characteristic of vertebrate GI PLA2s and lacks the C-terminal extension found in GII PLA2s but shares homology for both GI and GII PLA2s [19]. Comparison of the disulfide bond positions of the A. carciniopados sPLA2 with those of the human GI⁄ II ⁄ V ⁄ X PLA2 collection indicates that the sea anemone sPLA2 shares five con-served disulfide bonds with the vertebrate homologues, suggesting structural, and possibly functional, conser-vation across the phylogenetically distant organisms of Cnidaria and Mammalia (Fig. 3).
Conserved domain cd04707 of otoconins
Otoconins containing the conserved domain cd04707 were identified in a number of vertebrates, from fish to mammals, and two conserved domains within the N-terminal (OtoN) and C-terminal (OtoC) regions of the same collection were identified within the mature protein sequence (Tables 1 and S1). The human otoco-nin-90 is a 53 kDa protein, and the OtoN and OtoC domains are 37% identical and structurally closely related to the members of the GI⁄ II ⁄ V ⁄ X PLA2 collec-tion, suggesting common ancestry (Fig. 2). The human OtoN and OtoC domains are 36% and 34% identical, respectively, when compared with GIB PLA2. More-over, the disulfide bond patterns of human otoconin and GIB PLA2 are identical (Fig. 3). Both OtoN and OtoC domains contain a histidine residue within the conserved catalytic site. However, they are believed to be catalytically inactive as a result of mutations in the Ca2+-binding sites (Fig. 2), leading to loss of the usual Ca2+-binding residues in the OtoN domain, and in the OtoC domain the conserved second glycine of the mammalian GIB sPLA2s (Y-x-G-x-G) is replaced with a glutamic acid, although PLA2 activity remains to be investigated [29].
Conserved domain cd04704 of group III PLA2s Secreted PLA2s, containing the conserved domain cd04704 (bee venom-like PLA2), were identified in arthropods, reptiles and vertebrates, including humans.
H N
O. sativa GXIB PLA2
H N
S. pyogenes PLA2
A
B
Fig. 1. Alignment of the conserved domain cd04706 sequences of Streptococcus pyogenes sPLA2 (Spyo_06) and Oryza sativa GXIB PLA2(Osat2_06) (A) and the positions of the disulfide bonds (B). In panel A the four conserved cysteine (C) residues are indicated with ‘#’ and the conserved putative catalytic histidine (H) and asparagine (N) residues are highlighted in red and the Ca2+-binding residues tyrosine (Y), glycine (G) and aspartic acid (D) are highlighted in blue. The consensus symbols denote the degree of conservation: ‘*’, identical residues; ‘:’, conserved substitutions; and ‘.’, semiconser-vative substitutions. In (B) the conserved disulfide bonds C1-C2and C3-C4are red and green, respectively. H and N mark the histidine and asparagine residues of the catalytic sites, respectively. The solid horizontal line represents the mature peptide region.
The honeybee Apis mellifera venom GIII PLA2 shares considerable sequence identity with the members of the GI⁄ II ⁄ V ⁄ X PLA2collection (e.g. 44% identity with the human GIB PLA2) and is related in its 3D structure and catalytic mechanism to GI and GII PLA2s [11,17]. The backbone of the GIII PLA2 molecule contains the conserved Ca2+-binding loop with tryptophan, glycine and aspartic acid residues and the conserved catalytic histidine and aspartic acid residues (Fig. 4). Human GIII PLA2 is a 57 kDa protein in which cd04704 is localized in the middle part of the molecule and flanked by N- and C-terminal extensions [30]. The cd04704 of human GIII PLA2 displays features similar to the arthropod GIII PLA2s, including the Ca2+ -binding and catalytic site residues and also the 10 conserved cysteines that form five disulfide bonds at similar locations, and shares 44% sequence identity with honeybee venom GIII PLA2.
Conserved domain cd04705 of group III PLA2 Proteins containing the conserved domain cd04705 (similar to human group III PLA2) were found only in mammals (Table 1). The conserved domain cd04705 is localized in the C-terminal part of the GIII PLA2 mol-ecule and is structurally unrelated to cd04704 (Fig. 2). In contrast to cd04704, cd04705 is considered to be catalytically inactive and its function is unknown [30].
Conserved domain pfam09056 of PLA2s of bacteria, fungi and animals
The conserved domain pfam09056 (prokaryotic PLA2) was identified in the sPLA2s of Gram-positive and Gram-negative bacteria, protists, fungi and marine invertebrates of the phyla Cnidaria and Mollusca. Bacterial sPLA2s containing pfam09056 are markedly Fig. 2. Multiple sequence alignment of the conserved domains of human sPLA2s. HsapIB_25 (GIB PLA2), HsapIIA_25 (GIIA), HsapIIC_25 (GIIC), HsapIID_25 (GIID), HsapIIE_25 (GIIE), HsapIIF_25 (GIIF), HsapV_25 (GV) and HsapX_25 (GX) PLA2s contain the conserved domain cd00125 and belong to the structurally related GI⁄ II ⁄ V ⁄ X PLA2collection. HsapIII_04 and HsapIII_05 (GIII PLA2) represent cd04704 and cd04705, respectively. HsapGXIIA_51 and HsapGXIIB_51 (GXIIA and GXIIB PLA2s) contain pfam06951. HsapOtoN_07 and HsapOtoC_07 contain the N- and C-terminal sections of cd04707 of otoconin-90, respectively. The catalytic histidine (H), aspartic acid (D) and asparagine (N) residues are highlighted in red, and the Ca2+-binding amino-acid residues tyrosine (Y), glycine (G), histidine (H), phenylalanine (F), trypto-phan (W), proline (P), leucine (L) and aspartic acid (D) are highlighted in blue.
different in their molecular structure from the bacterial sPLA2s containing cd04706. Comparison of the conserved domains of Streptomyces violaceoruber (pfam09056) and S. pyogenes (cd04706) sPLA2s indicates that they share only 8% sequence identity. The catalytic motif of the sPLA2 of the Gram-positive bacterium S. viola-ceoruber [16,31] contains the conserved histidine and aspartic acid residues (Figs 2 and 5). The conserved
Ca2+-binding domain is absent and the binding of Ca2+is mediated via the aspartic acid and leucine resi-dues upstream of the catalytic site and by the aspartic acid residue adjacent to the catalytic histidine residue (Figs 2 and 5). There are four cysteines that form two putative disulfide bonds, including a bond that con-nects the catalytic and the Ca2+-binding sites (Fig. 5).
In addition to bacteria, sPLA2s containing pfam09056 were also identified in both unicellular and multicellular fungi. The sPLA2 of the fungus Tuber borchii [32] is a 23 kDa protein that contains two conserved disulfide bonds analogous to those of the sPLA2 of S. violaceoruber (Fig. 5), and the two conserved domains are 42% identical.
There are pfam09056-containing sPLA2s in aquatic invertebrates of the phylum Cnidaria, including the sea anemone N. vectensis and the hydrozoan Hydra magni-papillata. Cnidarian sPLA2s have the conserved cata-lytic histidine and aspartic acid residues but contain more numerous cysteine residues and putative disulfide bonds than the corresponding bacterial and fungal sPLA2s (Fig. 5).
Recently, expressed sequence tags (ESTs), coding for potential sPLA2s containing the conserved domain pfam09056, were identified in the protist Astrammina rara (phylum Foramifera) and the scallop Mizuhopec-ten yessoensis (phylum Mollusca) [33]. Previously, a related sPLA2 was isolated from the venom of another mollusc – the marine snail Conus magus – and classi-fied as GIX PLA2 [34] but no conserved domain was identified for this protein in the present study, proba-bly because of the incompleteness of its sequence. However, conserved histidine and aspartic acid active-site motifs are present in the pfam09056-containing sPLA2s of the scallop M. yessoensis and the sea anemone N. vectensis and in the GIX PLA2 of the
H D
A. carciniopados PLA2
H D
H. sapiens GV PLA2
H D
H. sapiens GIIA PLA2
H D H. sapiens GX PLA2 H H. sapiens otoconin D H D
H. sapiens GIB PLA2
Fig. 3. Comparison of the disulfide bond positions in the cnidarian Adamsia carciniopados and human GV, GIB, GIIA and GX PLA2s and otoconin-90. Five disulfide bonds are conserved from the cni-darian to the human. Histidine (H) and aspartic acid (D) are the active-site amino acid residues of the PLA2s, and the corresponding residues are present in otoconin. The basal line represents the mature peptide region.
Fig. 4. Alignment of the conserved domains cd04704 of the Apis mellifera GIII PLA2 (AmelIII_04) and human GIII PLA2 (Hsa-pIII_04). The Ca2+-binding residues tryptophan (W), glycine (G) and aspartic acid (D) are highlighted in blue, the catalytic histidine (H) and aspartic acid (D) are highlighted in red, and the conserved cys-teine (C) residues are indicated with ‘#’.
Fig. 5. Comparison of the conserved domains pfam09056 of Strep-tomyces violaceoruber (Svio_56), Tuber borchii (Tbor_56), Nemato-stella vectensis (Nvec11_56) and Mizuhopecten yessoensis (Myes_56) sPLA2s with the GIX PLA2of Conus magus. Four cyste-ine residues (C, marked with ‘#’) and the histidcyste-ine (H) and aspartic acid (D) residues of the active site (highlighted in red) are con-served. The putative Ca2+-binding residues aspartic acid (D), glycine (G) and leucine (L) are shown in blue. There are two putative disul-fide bonds between the conserved cysteines C1-C2and C3-C4.
snail C. magus (Fig. 5). Furthermore, the GIX PLA2 of C. magus shares 13–33% sequence identity with the bacterial, fungal and cnidarian pfam09056 PLA2s, but lower identity of 9–13% with the cd04706, cd04704 and cd00125 PLA2s.
Conserved domain pfam06951 of GXII PLA2s GXIIA PLA2 was first cloned from human [35]. In the present study, the conserved domain pfam06951 was identified in the GXII PLA2s of a large number of verte-brate and inverteverte-brate species, including the basal meta-zoans N. vectensis (Cnidaria), Trichoplax adhaerens (Placozoa) and Brachionus plicatus (Rotifera), as well as in the nonmetazoan eukaryotes (protists), such as the amoeba Naegleria gruberi (Heterolobosea), Monosiga ovata (Choanoflagellida), Euglena gracilis (Euglenozoa), Phytophthora infestans (Oomycetes), Thalassiosira pseudonana (Bacillariophyta), Capsaspora owczarzaki(Ichthyosporea), Chromera velia (Alveolata) and Thecamonas trahens (Apusozoa). Furthermore, pfam06951 was also identified in the sPLA2s of the mar-ine algae Micromonas (Viridiplantae) and the prokary-ote Planctomyces maris (Bacteria) (Table S1). These observations demonstrate that the conserved domain pfam06951 is widely distributed not only in the sPLA2s of higher animals but also in the sPLA2s of simple eukaryotes and prokaryotes. Sequence alignment of the GXIIA PLA2 conserved domains of organisms ranging from protists to mammals reveals high conservation of the Ca2+-binding and catalytic sites. The canonical
his-tidine catalytic site C-C-x-x-H-x-x-C motif is highly con-served. In the aspartic acid catalytic motif, the cysteine and aspartic acid residues are also conserved, with the exception of E. gracilis (Euglenozoa), Micromon-as pusilla (Viridiplantae) and P. infestans (Oomycetes), in which aspartic acid is replaced by glutamic acid and the first cysteine is absent in all the sequences (Fig. S1). Comparison of the human GXIIA PLA2 (pfam06951) with GIB PLA2 (cd00125) indicated 37% amino acid sequence identity.
Another gene product closely related to GXIIA PLA2 is GXIIB PLA2 [36]. Human GXIIB PLA2 shares 46% sequence identity with GXIIA PLA2. GXIIB PLA2 is a catalytically inactive protein as a result of the substitution of histidine with leucine in the catalytic site (Fig. 2).
Phylogenetic analysis of sPLA2s
Phylogenetic analysis of the conserved domains of sPLA2s, carried out with the maximum likelihood (ML) and neighbour-joining (NJ) methods, produced similar tree topologies, suggesting that the members of this protein family have a common and ancient evolu-tionary origin (Fig. 6). Two major sPLA2 groups – the cd-collection (which includes the cd00125, cd04704, cd04705, cd04706 and cd04707 of unicellular and mul-ticellular organisms) and the pfam-collection (which contains the sPLA2s with the annotated pfam06951 and pfam09056 domains) – exist and underwent dis-tinct trajectories during evolution.
Fig. 6. Consensus bootstrap phylogenetic tree of the sPLA2conserved domains. The tree was constructed with 53 taxa (see Table S1 for details) and using the NJ method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches, and bootstrap branches lower than 50 were collapsed. Acap_56, Ajellomyces capsulata (Fungi); AmelIII_04, Apis mellifera (Insecta); Aory2_56, Aspergillus oryzae (Fungi); Bbac_56, Bdellovibrio bacteriovorus (Deltaproteobacteria); BtauIII_05, Bos taurus (Mammalia); CfamIII_05, Canis familiaris (Mammalia); Cglo_56, Chaetomium globosum (Fungi); Cimm_56, Coccidioides immitis (Fungi); Crei_06, Chlamydomonas reinhardtii (Viridiplantae); Dcar_06, Dianthus caryophyllus (Viridiplantae); Dgeo_56, Deinococcus geothermalis (Deinococci); Dmel_25, Drosophila melanogaster (Insecta); DmelXIIA_51, Drosophila melanogaster (Insecta); Fsp_56, Frankia sp. (Actinobacteria); GgalOtoN_07, Gallus gallus (Aves); Hmag_56, Hydra magnipapillata (Cnidaria); HsapIII_05, Homo sapiens (Mammalia); HsapOtoN_07, Homo sapiens (Mammalia); HsapXIIA_51, Homo sapiens (Mammalia); Hsp_56, Helicosporium sp. (Fungi); HsusIII2_04, Heloderma suspectum (Anguimorpha); IpacXIIA_51, Ixodes pacificus (Arachnida); IscaIII_04, Ixodes scapularis (Arachnida); MdomXIIA_51, Monodelphis domestica (Mammalia); Medu_25, Mytilus edulis (Mollusca); Mext2_06, Methylobacterium extorquens (Alpha-proteobacteria): Mgri1_56, Magnaporthe grisea (Fungi); MmusIIA_25, Mus musculus (Mammalia); MmusIII_05, Mus musculus (Mammalia); MmusOtoC_07, Mus musculus (Mammalia); MmusOtoN_07, Mus musculus (Mammalia); MmusXIIA_51, Mus musculus (Mammalia); Mnod2_06, Methylobacterium nodulans (Alphaproteobacteria); Mrad_06, Methylobacterium radiotolerans (Alphaproteobacteria); Mtru_06, Medicago truncatula (Viridiplantae); Ntab 2_06, Nicotiana tabacum (Viridiplantae); Nvec10_56, Nematostella vectensis (Cnidaria); Nvec11_56, Nematostella vectensis (Cnidaria); Nvec12_65, Nematostella vectensis (Cnidaria); Nvec13_56, Nematostella vectensis (Cnidaria); OanaIB_25, Ornithorhynchus anatinus (Monotremata); OhanIA_25, Ophiophagus Hannah (Reptilia); Osat2_06, Oryza sativa (Viridiplantae); PperIII_04, Phlebotomus perniciosus (Insecta); Scoe1_56, Streptomyces coelicolor (Actinobacteria); Sequ_06, Streptococcus equi (Firmicutes); Sery_56, Saccharopolyspora erythraea (Actinobacteria); Spyo_06, Streptococcus pyogenes (Firmicutes); Svio_56, Streptomyces violaceoruber (Actino-bacteria); TgutXIIA_51, Taeniopygia guttata (Aves); TnigOtoC_07, Tetraodon nigroviridis (Teleostei); XlaeXIIA_51, Xenopus laevis (Amphibia); XtroIII_04, Xenopus tropicalis (Amphibia).
The sPLA2 forms of bacteria cluster within each PLA2s subgroup, suggesting that the eukaryote and prokaryote sPLA2 repertoire share a PLA2-like com-mon ancestor molecule, which remains to be identified. The cd-collection contains the majority of the sPLA2 sequences identified, and the clustering observed indi-cates that discrete relationships between the sPLA2 protein groups exist (Fig. 6). Members of the cd04704 and cd04705 groups seem to have evolved separately from the other cd-collection members, such as cd00125 and cd04707, and have only been identified in animals.
Discussion
PLA2 activity was first reported in canine pancreatic juice [37] and sPLA2s have now been isolated from a large number of snake and other venoms, and also from cells, tissues and body fluids of various unicellu-lar and multicelluunicellu-lar organisms [38–40]. Initially, sPLA2s were classified into two major groups of GI and GII PLA2s [41], but subsequent sequence homol-ogy and conserved disulfide bonds were observed within the other members of the GI⁄ II ⁄ V ⁄ X PLA2
col-HsapOtoN_07 TnigOtoC_07 Dmel_25 Mext2_06 Mrad_06 Spyo_06 Crei_06 Ntab2_06 Osat2_06 Dcar_06 Mtru_06 CfamIII_05 HsapIII_05 BtauIII_05 MmusIII_05 IscaIII_04 PperIII_04 XtroIII_04 HsusIII2_04 100 100 100 59 82 100 100 100 100 100 61 54 100 73 100 98 99 99 99 74 64 42 99 98 98 94 65 50 23 27 90 88 59 62 69 58 53 42 23 49 79 72 44 67 62 96 62 0.1 MmusOtoN_07 42 99 23 27 80 42 23 49 44 cd04707 cd00125 cd04706 Bacteria cd04706 Viridiplantae cd04705 cd04704 pfam06951 pfam09056 Bacteria pfam09056 Cnidaria pfam09056 Bacteria pfam09056 Fungi GgalOtoN_07 MmusOtoC_07 MmusIIA_25 OanaIB_25 OhanIA_25 Medu_25 Mnod2_06 Sequ_06 AmelIII_04 XlaeXIIA_51 TgutXIIA_51 HsapXIIA_51 MmusXIIA_51 MdomXIIA_51 IpacXIIA_51 DmelXIIA_51 Bbac_56 Nvec11_56 Nvec12_56 Nvec10_56 Hmag_56 Nvec13_56 Fsp_56 Dgeo_56 Scoe1_56 Svio_56 Mgri1_56 Sery_56 Aory2_56 Cimm_56 Cglo_56 Acap_56 Hsp_56 cd-collection pfam-collection
lection and the related protein otoconin [42]. This clas-sification has been expanded and refined over the past two decades as information from phylogenetically dis-tinct organisms has accumulated at an accelerating rate [1,2,8,23]. The recent systematic identification of the conserved domains of protein molecules [24,25] allows a novel classification of the sPLA2s (‘cd⁄ pfam classifi-cation’) based on the identification of the conserved domains, and a meaningful phylogenetic analysis of the whole range of sPLA2s.
The aim of the present study was to elucidate the origin and evolution of the sPLA2s. The enormous evolutionary span between the organisms expressing sPLA2s (ranging from prokaryotes to metazoan eukaryotes) by necessity introduces a degree of uncertainty in the comparison of protein sequences between such distant phyla. When PLA2 sequences containing conserved domains of different groups are compared, the molecular structure of a particular PLA2 reflects the divergent or convergent evolution of the conserved domain structure and the evolutionary history of the organism in question. In the present study, we investigated the structural variability of the conserved domains of sPLA2s in a wide range of organisms, from bacteria to mammals. A total of 358 conserved domains were identified among 333 sPLA2 sequences. In viruses, sPLA2-like proteins (GXIII PLA2) have also been reported but their molecular structure, including the conserved domain and the enzymatic activity, differ from those of sPLA2s [43] and thus were not included in the current analysis.
Two main distinct forms of sPLA2s were identified in bacteria: those containing cd04706 (bacteria⁄ plant-specific subfamily of PLA2) and those containing pfam09056 (prokaryotic⁄ fungal PLA2). Notably, no sPLA2s were identified in the Archaea. A marked difference between Bacteria and Archaea is that the predominant lipid constituents of the archaean mem-branes are prenyl ether lipids, whereas the bacterial membranes contain acyl ester lipids [44–46]. The phos-pholipid metabolism of bacteria is driven by phospho-lipases that specifically hydrolyze the acyl ester bonds but are incapable of hydrolyzing the prenyl ether lip-ids. In light of this observation, it is hypothesized that the sPLA2s of the higher organisms may have their evolutionary origin in Bacteria rather than in Archaea. Members of the sPLA2s are proposed to share a common ancestry and to have emerged early in evolu-tion, and several theories based upon their sequence similarities and predicted molecular structure have sug-gested that they evolved rapidly. The GI and GII sPLA2s of Elapidae and Crotalidae snake venoms,
respectively, have distinct molecular structures and it has been proposed that they share a common ancestor [22]. Snake venom sPLA2s are suggested to have evolved at an accelerated rate that has resulted in the presence of many variant PLA2molecules produced by the venom glands [40,47–49].
A recent example of the rapid evolution of sPLA2 genes is the bovine GIID PLA2. In cattle, five duplica-tions have been identified, while a single gene copy is present in the human and rodent genomes. The bovine GIID PLA2s are expressed in the mammary gland and up-regulated during the lactating period, and are sug-gested to participate in the innate immune response [50]. In human and mouse, GIIA, GIIC, GIID, GIIE, GIIF and GV PLA2 genes are also the result of gene-duplication events within the same chromosome. In human they are localized in chromosome 1, whereas GIB and GX genes map to chromosomes 12 and 16, respectively. However, comparisons between the gene homologues in human and mouse reveal that species-specific events may occur; for example, the human GIIC PLA2is a pseudogene, whereas the mouse homo-logue encodes an enzymatically active protein [51]. Another example of the functional diversity within the members of the GI⁄ II ⁄ V ⁄ X PLA2 collection are the human and mouse GIIA PLA2s, which are efficient bactericidal enzymes involved in the innate immune response, whereas the closely related digestive enzyme GIB PLA2 is only marginally bactericidal [6,52]. For example, in vitro assays demonstrated that Gram-posi-tive bacteria are killed by sPLA2s and that human GIIA PLA2 is highly potent in this respect, whereas GIB PLA2is of low efficacy [52].
In Cnidaria, a sister group of the vertebrate clade that diverged more than 500 million years ago [53,54], sPLA2s have been reported that structurally resemble the vertebrate GI and GII PLA2s [19– 21,28]. Our current observations indicate that a cni-darian sPLA2 [19] containing cd00125 has disulfide bonds at locations identical to those of the human GV PLA2 and conserved in other members of the GI⁄ II ⁄ V ⁄ X PLA2 collection. Group I PLA2s of ela-pid snake venoms have lost the ancestral pancreatic loop present in the cnidarian sPLA2 and also in the mammalian GIB PLA2. Such structural changes have resulted in the appearance of novel functions, includ-ing toxicity of the venom sPLA2s [55]. However, the number of cysteine residues and disulfide bonds of sPLA2s vary among closely related animals such as the sea anemones Adamsia carcinipados, Urtici-na crassicornis, Condylactis gigantea and N. vectensis [19–21,28,56]. The variation in the disulfide patterns and other structural features seems to preclude the
exact placement of these cnidarian sPLA2s in the currently recognized groups of the GI⁄ II ⁄ V ⁄ X PLA2 collection [19–21].
In the current study, homologues of the vertebrate GXII members with highly conserved domain regions were identified for the first time in unicellular organ-isms, and their function in such organisms remains to be established. In human, GXIIA PLA2is expressed in T lymphocytes and seems to be involved in the regula-tion of the immune response [57]. Human GXIIB PLA2was recently shown to be involved in the triglyc-eride metabolism in the liver [58] and is proposed to activate specific receptors that remain to be identified [36].
Life as we know it can be divided into the prokary-otic (cellular organisms that lack a nucleus) Domains of Bacteria (Eubacteria) and Archaea (Archaebacteria), and the Domain of Eukaryota (organisms consisting of nucleated cells, such as animals, plants and fungi) [44]. Prokaryotes are the oldest cellular life forms on Earth, dating back 3.5–4 billion years and predating eukaryotes by 1 billion years. The current phylogenetic analysis of the conserved domains of sPLA2s of the representatives of the major prokaryote and eukaryote taxa supports the hypothesis that the sPLA2s of the eukaryotes, including the Metazoa, Viridiplantae and Fungi, may have shared a common origin with their homologues in bacteria. Two sPLA2 groups (the cd-collection and the pfam-collection) emerged early in evolution and underwent distinct evolutionary trajecto-ries. Based upon the retrieved data and phylogenetic relatedness, a model for the evolution of the two sPLA2s group members is proposed centred around gene-duplication and gene-deletion events (Fig. 7A,B). While the two members of the pfam-collection are present in representatives of the Eubacteria and Ani-malia kingdoms and maintained throughout evolution (Fig. 7B), the cd-collection members seem to have mainly emerged in the Animalia kingdom (Fig. 7A) and their expansion may be associated with the gene duplications that are proposed to have contributed to the increase in organismal complexity during eukary-ote evolution [59]. The cd04706 preukary-otein members are exclusively found in Eubacteria and Plantae kingdoms and were lost from other life forms. Despite the lack of data from representatives of all clades and the fail-ure to identify homologues it can be hypothesized that in the kingdom Animalia, sPLA2s with the conserved domains cd00125 and cd04704 were the first members to emerge. Subsequently, several independent gene or genome-duplication events occurred and resulted in the emergence of two novel family members of verte-brate cd04704 and mammalian cd04705. The sequence
similarity observed between the vertebrate sPLA2s con-taining conserved domains cd04707 and cd00125 and also the mammalian cd04705 with cd04704 suggests that they have a shared common origin and that cd04707 emerged from a gene-duplication event of the cd00125-like ancestral gene at the time of vertebrate emergence and that cd04705 resulted from a later gene-duplication event of the cd04704-like ancestral gene precursor within the mammalian lineage (Fig. 7A). In contrast, only fungi, protists and aquatic invertebrates, such as those of the phyla Cnidaria and Mollusca, have acquired pfam09056-containing sPLA2s and the homologue gene seems to have been deleted from Protostomes and Deuterostome genomes (Fig. 7B). Deuterostomia Protostomia Cnidaria ANIMALIA FUNGI PLANTAE PROTISTA EUBACTERIA
Universal common ancestor
ARCHAEBACTERIA Placozoa ? cd04706 cd04706 ? ? cd04707 cd00125 cd04705 cd04704 cd00125 cd04704 cd04704/ cd00125 cd00125 Emergence of cd04707 and cd04705 Gene duplication Emergence of cd04704 and cd00125 (vertebrata) (mammalia) A Deuterostomia Protostomia Cnidaria ANIMALIA FUNGI PLANTAE PROTISTA EUBACTERIA
Universal common ancestor
ARCHAEBACTERIA pfam09056 pfam06951 pfam06951 pfam06951 pfam06951 pfam06951 pfam06951 pfam09056/ pfam06951 pfam09056 Deletion of pfam09056 pfam09056 pfam06951 Deletion of pfam09056 Placozoa ? pfam09056 B
Fig. 7. Proposed evolution of sPLA2s in the six Kingdoms of life (Animalia, Fungi, Plantae, Protista, Archaeobacteria and Eubacteria). Models were constructed using the retrieved data obtained and phylogenetic relationships. (A) Hypothetical evolution of the sPLA2 cd-collection members. (B) Hypothetical evolution of the pfam-col-lection members. Dashed arrows indicate unclear evolutionary trajectories and ‘?’ not yet identified.
The present analysis is based on the highly con-served peptide motifs directly involved in the catalytic function of sPLA2s, including the Ca2+-binding site and the catalytic centre. However, the functional roles of the surrounding domains are at present less well understood (e.g. the functions of the domains flanking the central catalytically active cd04704 of mammalian GIII PLA2s are unknown) [3] and their study may pro-vide novel insight into the catalytic and noncatalytic functions of sPLA2s. For instance, binding of sPLA2s to specific cellular receptors is independent of their cat-alytic activity, and the protein domains involved in the receptor activation are not yet fully resolved [4]. Other examples are the toxicity of some snake venom PLA2s, which does not correlate with their catalytic activity [8], and the bactericidal effects of sPLA2s lacking cata-lytic activity [60].
It is concluded that the sPLA2s of eukaryotes share their evolutionary origin with two distinct types of bacterial sPLA2s. Their evolution and prevalence in genomes seems to be related to the functional con-straints of phospholipid metabolism which is a funda-mental and conserved process in organisms. Although relatively little is known about the function of prokay-otic sPLA2s, the large number of distinct sPLA2 iso-forms in metazoans reflects the wide variation of substrate types encountered in the extracellular envi-ronment where the enzyme plays many important roles in nutrition, reproduction and immunity.
Material and methods
Database mining and data collection
Secreted PLA2 sequences were retrieved from the publicly
available protein databases of NCBI (http://www.ncbi.nlm.-nih.gov) and Swiss-Prot (http://www.uniprot.org) using the Basic Local Alignment Search Tool (BLASTp) algorithm [61] and default settings. Database searches were performed using the peptide sequences of the human GIB PLA2
(P04054), honeybee venom GIII PLA2 (P00630), human
GXIIA PLA2(Q9BZM1), O. sativa GXIB PLA2(Q9XG81)
and S. violaceoruber PLA2 (Q6UV28). In addition, PLA2
and PLA2-like protein sequences were identified in the
Microbial and Eukaryotic Genome database (http:// www.ncbi.nlm.nih.gov/genome) following a similar strategy. Searches were also performed on nucleotide database data and covered all completed genomes in the NCBI genome database (1359 bacterial, 79 archaeal and 231 eukaryotic genomes; October 2010 release), and also available EST data using the tBLASTn and sequence matches with an e-value of < 10 were retrieved and their sequence analysed. The deduced protein sequences were obtained using the BCM Search Launcher (http://searchlauncher.bcm.tmc.edu/
seq-util/Options/sixframe.html) and compared with avail-able homologue data.
In silico sequence annotations
The conserved domains of sPLA2were identified using the
NCBI Conserved Domains Database CDD-27036 PSSMs (http://www.ncbi.nlm.nih.gov/cdd) annotation by using the sequences identified in this study as queries. The result includes an alignment between the query and the search model consensus sequence, the expected-value for the align-ment, the identity (name) of the conserved domain and the location of the conserved domain in the query sequence [25]. Histidine active-site and aspartic acid active-site pro-tein motifs were identified based on the PROSITE database (http://au.expasy.org/prosite) pattern annotation. The local-ization of disulfide bonds was retrieved from the Swiss-Prot database and from published data.
Sequence comparisons and phylogenetic analysis
Pairwise and multiple protein sequence alignments were carried out using the ClustalW2 program [62] available from EBI (http://www.ebi.ac.uk/Tools/msa/clustalw2) and the default parameters. The percentage of sequence simi-larities (based on the observed substitutions of one amino acid for another in homologous proteins) and identities between the sPLA2s were calculated based upon protein
alignments using the GeneDoc interface (http:// www.psc.edu/biomed/genedoc). Phylogenetic analysis of the conserved domain sequences of bacterial, fungal and metazoan PLA2s was performed using 53 taxa
representa-tives. The protein alignment produced was submitted to PROTTEST analysis (http://darwin.uvigo.es/software/prot-test.html) to select the best model of protein evolution that fits the data set [63]. Phylogenetic analyses were con-ducted using the NJ [64] and ML methods, and reliability for internal branching was assessed using the bootstrap method [65]. NJ analysis was performed using MEGA4 programme [66] and the p-distance amino acid model with 1000 bootstrap replicates. All positions containing align-ment gaps and missing data were eliminated (pairwise deletion option) and a total of 215 positions were ana-lyzed in the final data set. The ML tree (PhyML, v3.0 aLRT) [67] was constructed in Phylogeny.fr web interface (http://phylogeny.lirmm.fr/phylo_cgi/index.cgi). The WAG substitution model was selected assuming an estimated proportion of invariant sites (of 0.167) and four gamma-distributed rate categories to account for rate het-erogeneity across sites. The gamma shape parameter was estimated directly from the data (c = 1.804) and analysis was performed using 100 bootstrap replicates. Graphical representation and edition of the phylogenetic tree were performed with treedyn (v198.3, http://www.treedyn.org).
Both methods produced similar tree topologies and the NJ bootstrap consensus tree was selected and taxa clades with support values < 50% collapsed.
Acknowledgements
The authors thank Deborah Power for critical reading of the manuscript and anonymous referees for valuable suggestions. Supported by the Research Fund of Turku University Hospital and the Portuguese National Science Foundation (FCT)⁄ CCMAR pluriannual grant.
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Supporting information
The following supplementary material is available: Fig. S1. Alignment of the conserved domain pfam06951 sequences of 20 GXIIA PLA2s.
Table S1. Conserved domains of 333 prokaryotic and eukaryotic secreted phospholipases A2.
This supplementary material can be found in the online version of this article.
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